Method and System for Detection of Natural High Intensity Sweeteners that Contain Hydroxyl Groups

ABSTRACT

The present invention provides a hapten derivative and conjugate of a natural high intensity sweetener containing hydroxyl groups. The conjugate can be used to produce antibodies specific against the natural high intensity sweetener. The present invention further provides a kit and method for detecting and quantifying the natural high intensity sweetener in a sample.

RELATED APPLICATIONS

The present invention is a continuation application of U.S. patentapplication Ser. No. 15/242,618 filed Aug. 22, 2016, which is adivisional application of U.S. patent application Ser. No. 14/346,967filed Mar. 25, 2014, which is a national phase application ofInternational patent application no. PCT/MY2012/000138, filed Jun. 22,2012, which claims the benefit of International patent application no.PCT/MY2011/000209, filed Sep. 27, 2011, the disclosure of which isincorporated in its entirety.

FIELD OF INVENTION

The present invention relates to method and system for the detection ofsteviol glycosides.

BACKGROUND OF THE INVENTION

High intensity sweeteners possess a sweetness level many times exceedingthat of sucrose. They are widely used in diet and calorie-reduced foodas being essentially non-caloric, not affecting blood glucose level, andprovide little or no nutritive value. In contrast, natural caloricsweeteners such as sucrose, fructose, and glucose are not suitable fordiet and calorie-reduced food for being highly caloric, even though theyprovide the most desirable taste to consumers.

Stevia rebaudiana Bertoni is a perennial shrub of the Asteraceae(Compositae) family native to certain regions of South America. Theleaves of the plant contain from 10 to 20% of diterpene glycosides,which are around 150 to 450 times sweeter than sugar. The leaves havebeen traditionally used for hundreds of years in Paraguay and Brazil tosweeten local teas and medicines.

At present there are more than 230 Stevia species with significantsweetening properties. The plant has been successfully grown under awide range of conditions from its native subtropics to the cold northernlatitudes.

The extract of Stevia rebaudiana plant contains a mixture of differentsweet diterpene glycosides, which have a single base—steviol and differby the presence of carbohydrate residues at positions C₁₃ and C₁₉. Theseglycosides accumulate in Stevia leaves and compose approximately 10%-20%of the total dry weight. The steviol glycosides include Dulcoside A,Rebaudiosides A, B, C, D, E, and F, steviolbioside, and Rubusoside.Typically, on a dry weight basis, the four major glycosides found in theleaves of Stevia are Dulcoside A (0.3%), Rebaudioside C (0.6-1.0%),Rebaudioside A (3.8%) and Stevioside (9.1%). Among steviol glycosidesonly Stevioside and Rebaudioside A are available in commercial scale.

Steviol glycosides have zero calories and can be used wherever sugar isused. They are ideal for diabetic and low calorie diets. In addition,the sweet steviol glycosides possess functional and sensory propertiessuperior to those of many high potency sweeteners.

Rebaudioside D (CAS No: 63279-13-0) is one of the sweet glycosides foundin Stevia rebaudiana.

Studies show that highly purified forms of Rebaudioside D possess verydesirable taste profile, almost lacking bitterness, lingering licoriceaftertaste typical for other Steviol glycosides. These propertiesmultiply the significance of Rebaudioside D and attract great interestfor methods of preparation of highly purified forms of Rebaudioside D.

It has to be noted that in commercially available Stevia rebaudianavarieties rebaudisoide D content is very low. Generally on a dry weightbasis the leaves of Stevia rebaudiana contain 0-0.1% rebaudioside D.Therefore there's a need of developing new varieties of Steviarebaudiana with higher content of rebaudioside D. One of the keyconditions for such development work is the availability of simple andhigh throughput testing methodologies, which can be applied on a largenumber of samples.

On the other hand, the testing methodologies which exist today forsteviol glycosides' analysis employ very sophisticated and expensiveHPLC techniques with very low throughput. These methods, although robustand well established, are time consuming, expensive, require specialisedtechnicians and instrumentation, and the number of samples that can beprocessed daily is small. In addition, the amounts of chemicals andtoxic solvents that are used often have a high environmental risk.

These disadvantages clearly show the need for developing fast,easy-to-use, robust, sensitive and cost-effective techniques for highthroughput analysis of steviol glycosides, particularly rebaudioside Din various matrices, including plant biomass.

SUMMARY OF THE INVENTION

One aspect of the present invention provides a hapten derivative of anatural high intensity sweetener containing hydroxyl groups. In oneembodiment, the hapten derivative has the structure selected from thegroup consisting of:

wherein the hapten derivative is used for further making conjugates forbeing used as an immunogen for generating antibodies specific for thenatural high intensity sweetener or a reagent for detecting the naturalhigh intensity sweetener.

In another embodiment of the hapten derivative, the natural highintensity sweetener is selected from the group consisting of DulcosideA, Rebaudiosides A, B, C, D, E, and F, Steviolbioside and Rubusoside.

In another embodiment of the hapten derivative, the R2 is formed byreacting the natural high intensity sweetener with sodium periodate.

In another embodiment of the hapten derivative, the R3 is formed byreacting the natural high intensity sweetener withN,N′-carbonyldiimidazole.

In another embodiment of the hapten derivative, the R4 is formed byreacting the natural high intensity sweetener with3-aminopropyltrimethoxysilane.

In another embodiment of the hapten derivative, the R5 is formed byreacting the natural high intensity sweetener with(3-propilisocianate)triethoxysilane.

Another aspect of the present invention provides a conjugate of anatural high intensity sweetener containing hydroxyl groups. In oneembodiment, the conjugate has the structure of formula (1) and (2). Bothstructures are a simplified representation of the possible conjugates,as all the sugar rings are susceptible of suffering that chemicalmodification, wherein X is a linker chain comprising 1-8 carbon orhetero atoms; Y is selected from the group consisting of —CO, —NH₂ and—NCO; and Z is an antigenic carrier or a label; and wherein theconjugate is used as an immunogen for generating antibodies specific forthe natural high intensity sweetener or a reagent for detecting thenatural high intensity sweetener.

In another embodiment of the conjugate, the linker chain is substitutedor unsubstituted.

In another embodiment of the conjugate, the linker is straight orbranched.

In another embodiment of the conjugate, X is —Si(OMe)₂—CH₂—CH₂—CH₂.

In another embodiment of the conjugate, Y is —NH₂.

In another embodiment of the conjugate, the label is selected from thegroup consisting of radioactive isotopes, enzymes, enzyme fragments,enzyme substrates, enzyme inhibitors, coenzymes, catalysts,fluorophores, dyes, chemiluminescers, luminescers, sensitizers,non-magnetic or magnetic particles, solid supports, liposomes, ligands,receptors and hapten radioactive isotopes.

In another embodiment of the conjugate, the natural high intensitysweetener is selected from the group consisting of Dulcoside A,Rebaudiosides A, B, C, D, E, and F, steviolbioside, and Rubusoside.

Another aspect of the present invention provides a kit for detecting anatural high intensity sweetener containing hydroxyl groups. In oneembodiment, the kit comprises a conjugate of the natural high intensitysweetener with a detectable label as a tracer; an antibody specificagainst the natural high intensity sweetener; and optionally adetectable enzymatic substrate if the detectable label is an enzyme;wherein the antibody is coated onto a support; and wherein when thesample containing the natural high intensity sweetener is contacted withthe tracer first to form a mixture and the mixture is then contactedwith the coated antibody, the quantity of the tracer bound to the coatedantibody is inversely correlating with the concentration of the naturalhigh intensity sweetener in the sample.

In another embodiment of the kit, the detectable label is selected fromthe group consisting of radioactive isotopes, enzymes, enzyme fragments,enzyme substrates, enzyme inhibitors, coenzymes, catalysts,fluorophores, dyes, chemiluminescers, luminescers, sensitizers,non-magnetic or magnetic particles, solid supports, liposomes, ligands,receptors and hapten radioactive isotopes.

In another embodiment of the kit, the natural high intensity sweeteneris selected from the group consisting of Dulcoside A, Rebaudiosides A,B, C, D, E, and F, steviolbioside, and Rubusoside.

Another aspect of the present invention provides a method for detectinga natural high intensity sweetener containing hydroxyl groups in asample. In one embodiment, the method comprises providing a conjugate ofthe natural high intensity sweetener with a detectable label as atracer; contacting the tracer with the sample to form a mixture;providing an antibody specific against the natural high intensitysweetener, wherein the antibody is coated onto a solid support;contacting the mixture with the coated antibody; and optionallyproviding a detectable enzymatic substrate if the detectable label is anenzyme; wherein the quantity of the tracer bound to the coated antibodyis inversely correlating with the concentration of the natural highintensity sweetener in the sample.

Another aspect of the present invention provides a kit for detecting anatural high intensity sweetener containing hydroxyl groups. In oneembodiment, the kit comprises a conjugate of the natural high intensitysweetener; an antibody specific against the natural high intensitysweetener, wherein the antibody is conjugated with a detectable label;and optionally a detectable enzymatic substrate if the detectable labelis an enzyme; wherein the conjugate is coated onto a support; andwherein when the sample containing the natural high intensity sweeteneris contacted with the antibody first to form a mixture and the mixtureis then contacted with the coated conjugate, the quantity of theantibody bound to the coated conjugate is inversely correlating with theconcentration of the natural high intensity sweetener in the sample.

Another aspect of the present invention provides a method for detectinga natural high intensity sweetener containing hydroxyl groups. In oneembodiment, the method comprises providing a conjugate of the naturalhigh intensity sweetener that is coated onto a solid support; providingan antibody specific against the natural high intensity sweetener,wherein the antibody is conjugated with a detectable label; contactingthe antibody with a detectable label with the sample to form a mixtureallowing the natural high intensity sweetener in the sample to reactwith the antibody first; contacting the mixture with the coatedconjugate; and optionally providing a detectable enzymatic substrate ifthe detectable label is an enzyme; wherein the quantity of the antibodybound to the coated conjugate is inversely correlating with theconcentration of the natural high intensity sweetener in the sample.

Additional features of the present invention will become apparent tothose skilled in the art upon consideration of the following detaileddescription of preferred embodiments exemplifying the best mode ofcarrying out the invention as presently perceived.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying drawings are included to provide a furtherunderstanding of the invention. The drawings illustrate embodiments ofthe invention and together with the description serve to explain theprinciples of the embodiments of the invention. Preferred embodimentsaccording to the present invention will now be described with referenceto the Figures, in which like reference numerals denote like elements.

FIG. 1 shows the structure of Rebaudioside D (CAS No: 63279-13-0),designated as R₁ hapten.

FIG. 2 shows the structures (Formula 1 and 2) of an illustrativeRebaudioside D derivative of the present invention.

FIG. 3 shows the structure of R₂ hapten.

FIG. 4 shows the structure of R₃ hapten.

FIG. 5 shows the structure of R₄ hapten.

FIG. 6 shows the structure of R₅ hapten.

FIG. 7 shows immunoassay formats.

FIG. 8 shows a competitive calibration curve for Rebaudioside D underoptimized conditions (n=20).

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of certain embodiments of the invention.

Throughout this application, where publications are referenced, thedisclosures of these publications are hereby incorporated by reference,in their entireties, into this application in order to more fullydescribe the state of art to which this invention pertains.

Immunoassays (IAs) can analyze and monitor many targets at desirable orregulatory levels without any or minimal sample preparation. Thestrength of IAs lies in their capability of screening of a large numberof samples within a short time, being a valuable supplement to otheranalytical methods.

Immunoassays have been widely and successfully used in clinicalchemistry and veterinary drug registration for many years. Extensiveliterature is available which indicates the potential of immunoassays asfast, reliable and cost efficient methods in residue analysis ofdifferent analytes. In contrast, the development of immunoassays byindustries and their acceptance by registration agencies as analyticalmethods for the monitoring of agrochemical residues is scarce.

All immunoassays rely on the interaction between an antibody and atarget analyte. Antibodies are produced in response to an immunogen by acomplex mechanism. Approximately 90% of the developed immunoassays forresidue analyses use the Enzyme-Linked Immunosorbent Assay (ELISA). Inthis technique the analyte from the sample and a known amount ofenzyme-tagged analyte compete for a limited number of antibody bindingsites. Quantification is achieved by comparing the signal generated byan unknown sample with a standard curve.

In water, ELISA assays are generally used as fast stand-alone methods.In solid matrices they usually serve as detectors in sample extractswith or without clean-up step. In this matter, immunoassays are able todetect low concentrations in many samples in a short time, and often donot require laborious extraction or cleanup steps, making itsparticularly suitable for screening purposes.

The present invention attempted to develop a method and system fordetection and quantification of natural high intensity sweeteners thatcontain hydroxyl groups. For example, the natural high intensitysweeteners from Stevia includes Dulcoside A, Rebaudiosides A, B, C, D,E, and F, steviolbioside, and Rubusoside. The exemplary natural highintensity sweetener is Rebaudioside D (RebD). It is to be noted thatRebD is used for the sole purpose to illustrate the principles of thepresent invention; by no means, the present invention is limited toRebD.

RebD (CAS No: 63279-13-0) is one of the sweet glycosides found in Steviarebaudiana, and has the structure as shown in FIG. 1.

RebD itself is not immunogenic (hapten). As is a hapten, RebD needs tobe conjugated to a protein carrier so that RebD-specific antibodies canbe produced. The inventors of the present invention found that RebDcould not be conjugated readily to a protein carrier. After extensiveexploration, the inventors of the present invention discovered that RebDcould be first activated through its hydroxyl group (RebD derivativehaptens) and then the activated RebD derivatives could be readilyconjugated with a protein carrier. The RebD derivative-proteinconjugates are able to produce RebD-specific antibodies. Then, theRebD-specific antibodies can be used to develop methods and systems forRebD detection and quantification.

“Haptens” are partial or incompletes antigens. They are usuallyprotein-free substances, mostly of low molecular weight, which alone arenot capable of stimulating antibody formation, but which do react withantibodies. Hapten-specific antibodies may be formed by coupling ahapten to a high molecular weight antigenic carrier (macromolecule) andthen injecting this coupled product, i.e., immunogen, into a human oranimal subject. In the present invention, RebD is designated as R₁hapten, and RebD derivative haptens are designated as haptens R₂-R₅ thatwere synthesized by chemical derivatisation of RebD (detailed discussionof haptens R₂-R₅ is provided hereinafter).

In one embodiment, the present invention provides a hapten-carrierconjugate having the following structure (Formula 2) (FIG. 2):

X is a linker chain comprising 1-8 carbon or hetero atoms, wherein thelinker chain may be substituted or unsubstituted and may be straight orbranched. Y is selected from the group consisting of —CO, —NH₂, andNCO—; and Z is an antigenic carrier or a label.

In one particular illustrative embodiment, X is —Si(OMe)₂—CH₂—CH₂—CH₂, Yis —NH₂, and Z is the antigenic carrier. Antibodies produced using suchcompounds and immunoassay kits using the antibodies are also provided.

The phrase “antibody capable of specifically binding Rebaudioside D” asused herein refers to an antibody that is of capacity to react with atleast one epitope within RebD in a true antibody-antigen reactionforming an antibody-antigen complex, as opposed to non-specificinteraction.

The term “analog” or “derivative” refers to a chemical compound ormolecule made from a Rebaudioside D parent compound or molecule by oneor more chemical reactions.

An “activated hapten” refers to a hapten derivative that has beenprovided with an available site for reaction, such as by the attachmentof a linking group, for synthesizing a hapten derivative conjugate.

As used herein, a “linking group” or “linker” refers to a portion of achemical structure that connects two or more substructures such ashaptens, carriers, immunogens, labels, tracers, or other linkers. Alinking group has at least one uninterrupted chain of atoms other thanhydrogen (or other monovalent atoms), extending between thesubstructures. The atoms of a linking group and the atoms of a chainwithin a linking group are themselves connected by chemical bonds.Linkers may be straight or branched, saturated or unsaturated carbonchains. They may also include one or more heteroatoms within the chainor at termini of the chains. By “heteroatoms” it is meant atoms otherthan carbon atoms, illustratively oxygen, nitrogen and silicon, notlimited to others as sulfur or phosphorus. The linking group may alsoinclude cyclic or aromatic groups as part of the chain or as asubstitution on one of the atoms in the chain. The number of atoms in alinking group or linker is determined by counting the atoms other thanhydrogen. The number of atoms in a chain within a linking group isdetermined by counting the number of atoms other than hydrogen along theshortest route between the substructures being connected. Linking groupsmay be used to activate a hapten, e.g. provide an available site on ahapten for synthesizing a conjugate of a hapten with a label or carrier.

The terms “immunogen” and “immunogenic” as used herein refer tosubstances capable of producing or generating a strong immune responsein a mammalian organism. Sometimes used synonymously with antigen.

A “carrier” or “immunogenic carrier,” as the terms are used herein, isan immunogenic substance, commonly a protein, that can join with ahapten, thereby enabling the hapten to induce an immune response andelicit the production of antibodies that can bind specifically with theantigen (hapten). Carrier substances include proteins, glycoproteins,complex polysaccharides, particles and nucleic acids that are recognizedas foreign and thereby elicit an immunologic response from the host.Various proteins may be employed as a poly (amino acid) immunogeniccarrier. These proteins include albumins and serum proteins, e.g.,globulins, ocular lens proteins, lipoproteins, etc. Illustrativeproteins include bovine serum albumin (BSA), keyhole limpet hemocyanin(KLH), egg ovalbumin (OVA), bovine gamma-globulin (BGG), etc.

A “label”, “detector molecule”, or “tracer” is any molecule thatproduces, or can be induced to produce, a detectable signal. The labelcan be conjugated to an analyte, an immunogen, an antibody;illustratively the antibody produced in response to the antigeniccompound or a secondary antibody having specificity against first oneantibody, or to another molecule such as a receptor or a molecule thatcan bind to a receptor such as a ligand, particularly a hapten.Non-limiting examples of labels include radioactive isotopes, enzymes,enzyme fragments, enzyme substrates, enzyme inhibitors, coenzymes,catalysts, fluorophores, dyes, chemiluminescers, luminescers,sensitizers, non-magnetic or magnetic particles, solid supports,liposomes, ligands, receptors and hapten radioactive isotopes.

The term “antigenic compound” as used herein is a compound used toproduce an immune response. Illustratively, the antigenic compound is ahapten, for example Rebaudioside D, linked to an immunogenic carrier.The antigenic compound is used to generate the desired antibodies.

The term “labeled competitor” or “conjugate” as used herein is formingby covalently coupling two molecules together such as a hapten with aprotein or a detectable label or tracer capable of specific binding toantibodies having specificity for Rebaudioside D. Illustratively, themolecule is Rebaudioside D or a derivative or analyte thereof.

A “substrate” is a chemical that specifically reacts with an enzyme.

The term “sample” includes, but not limited to, any quantity of asubstance from plant biomass or foods.

Accordingly, developed assays may provide sensitivity across a broaderrange of Rebaudioside D concentrations.

Numerous quantitative immunoassay formats for detecting a hapten orother small molecule in a sample are known. An assay method forRebaudioside D illustratively includes combining the sample with ananti-rebaudioside D antibody and detecting the amount of theanti-rebaudioside D antibody-rebaudioside D complex, as indicative ofthe amount of Rebaudioside D in the sample.

Illustrative immunoassays employ polyclonal antibodies, non-limiting formonoclonal antibodies, with appropriate sensitivity and specificity toRebaudioside D to provide information about Rebaudioside Dconcentrations, statistically comparable to that obtained throughanalytical methods such as HPLC. Such immunoassays illustratively areuseful in monitoring levels of the sweet glycosides in plant biomass.

Designing an immunoassay for the detection of a small molecule such asRebaudioside D can be a challenge. Such small molecules often lackantigenicity, making it difficult to generate antibodies. To increasethe immunogenicity, larger antigenic compounds, illustratively proteinsor polypeptides, including but not limited to bovine serum albumin,ovalbumin, keyhole limpet hemocyanin, and the like, are conjugated tothe analyte of interest. Further, detection of the target analyte in animmunoassay generally requires the use of a detectable label conjugatedto an antibody, an analyte, or analyte analog.

Immunogens may be made by coupling Rebaudioside D to an antigeniccarrier protein through a spacer arm. However, it has been found that anextended linker between the antigenic carrier leads to the production ofmore sensitive antibodies. Without being bound to any particular theory,presumably, the longer linker provides for a more accessible epitope,resulting in increased specificity of the antibody for Rebaudioside D.

BSA-hapten and KLH-hapten, were used as immunogens, HRP-hapten, goatanti-rabbit immunoglobulins-HRP (GAR-HRP) as enzymatic tracer andHb-hapten, OVA-hapten and BSA-hapten as coating conjugates.

It is understood that any combination of antibodies produced using theabove-described antigenic compounds and the above-described labeledcompetitors may be used in competitive assays, the choice of whichdepends on the specific assay and desired sensitivity.

Traditionally, detection of low-molecular-mass analytes (haptens) suchas Rebaudioside D in solution must employ competitive immunoassayformats. There are two different competitive formats available, (1) withimmobilized antibody (FIG. 7a ) and (2) with immobilized coatingconjugate (FIG. 7b ) onto the surface of each micro-well in a 96-wellmicro plate and held in place via non-covalent bonds between thehydrophobic regions of the protein and the non-polar plastic surface. Informat (1), analyte and labeled analyte (tracer) compete for the freeantibody binding sites. After removal of unbound reactants the boundtracer yields a signal. The format (2) employs an immobilizedhapten-carrier conjugate on the solid phase to which analyte andantibody are added. Antibody binds to the free analyte or to theimmobilized hapten in certain ration of the reactants concentration. Ifa labeled antibody is used, the amount of antibody bound to the solidphase can be directly determined after a washing step. Alternatively, asecondary labeled antibody (such as GAR-HRP) may be used to detect thebound antibody. In these competitive immunoassay formats, the signal isinversely proportional to the amount of free analyte in the sample. Inenzyme labeled competitive immunoassay, separation of unbound reagentfrom bound reagent is needed, i.e. heterogeneous assay is performed.

The derivatives, antibodies, immunogens, and/or other conjugatesdescribed herein are also suitable for use with any of a number of otherhomogeneous and heterogeneous immunoassays with a range of detectionsystems. The examples presented herein are not intended to be limiting.

Thus, the present invention provides Rebaudioside D derivatives that areuseful for the preparation of immunogens and conjugates for use inimmunoassays for the detection of Rebaudioside D. By coupling aRebaudioside D analog according to the present invention to animmunogenic carrier material, polyclonal or monoclonal antibodies can beproduced and isolated, which are useful reagents for immunoassays forthe detection of Rebaudioside D.

The term “polyclonal antibodies” as used herein is a population ofantibodies with various selectivities and affinities produced by manyclones of antibody-producing cells.

The term “monoclonal antibodies” as used herein refer to a homogeneousantibody population, possessing identical selectivity and affinityproduced by a single clone of antibody-producing cells.

Illustrative Rebaudioside D immunoassays employ anti-Rebaudioside Dantibodies that can be either polyclonal or monoclonal. In illustrativecompetitive immunoassays, the antibody preparation used is induced by animmunogen described herein is formulated in an aqueous solution such asbuffer, and the like or provided in an adjuvant or similar composition.The induced antibodies can be tested to determine specificity forRebaudioside D.

The following examples are provided for the sole purpose of illustratingthe principles of the present invention; it by no means shall beinterpreted for limiting the scope of the present invention.

EXAMPLE I Synthesis of Modified RebDs as RebD Haptens, Labeled RebDHaptens as RebD Competitors, and RebD Hapten-Protein Conjugates I.1.RebD Hapten R₂ and Protein-R₂ Conjugate

Rebaudioside D (5 mg) was dissolved in 1.2 mL of distilled water. Then,0.1 M sodium periodate in 10 mM PBS (300 mL) was added, and the reactionmixture was incubated for 20 minutes at room temperature, obtaining theRebD hapten R₂ (FIG. 3).

For synthesis of protein-R₂ conjugates, proteins (10 mg for BSA and KLHor 3 mg in the case of HRP) were dissolved in 1 mL of carbonate bufferand added to the reaction mixture containing the RebD hapten R₂,incubating with stirring for 2 h at room temperature. The mixture wascooled at 4° C. and the formed imines were quenched with 100 mL ofsodium borhydrure (4 mg/mL in distilled water) for 2 hours at 4° C. Theconjugates were purified through a molecular exclusion column(Desalting, Pierce) and protein concentration was determined by Bradfordmethod or by direct measurement of absorbance at 404 nm in the case ofthe HRP enzyme.

I.2. RebD Hapten R₃ and Protein-R₃ Conjugate

N,N′-carbonyldiimidazole (0.25 mmol, 41 mg) was added into a solution ofRebaudioside D (0.042 mmol, 47 mg) in anhydrous dimethylformamide (0.5mL) under inert atmosphere at 0° C., incubating for 3.5 hours, obtainingRebD hapten R₃ (FIG. 4) After this, 0.25 mL of the reaction mixture wasadded to a protein solution (15 mg BSA or 10 mg of KLH in 1.8 mL ofcarbonate buffer) and incubated at 4° C. for 48 hours. The conjugateswere purified and the protein concentration was measured as describedabove.

I.3. RebD Hapten R₄ and Protein-R₄ Conjugate

Rebaudioside D (50 mg) was silanised by vapour phase reaction with3-aminopropyltrimethoxysilane in a chamber, at room temperature for 3 h,obtaining RebD hapten R₄ (FIG. 5). The presence of primary amino groupswas verified by Sarin method. For the synthesis of protein-R4conjugates, a solution of protein (10 mg of KLH, BSA, OVA and Hb in 5 mLof 10 mM PBS, or 3 mg of HRP in 1.5 mL of PBS) was provided. Five mg ofRebD hapten R₄ and glutaraldehyde at 25% (200 mL) were added to theprotein solution, leaving the reaction for 4 hours at 4° C. Then, toquench the imines formed, 50 mg of sodium borhydrure was added and leftfor 1 h at 4° C. These conjugates were used directly withoutpurification.

I.4. RebD Hapten R₅ and Protein-R₅ Conjugate

50 mg of Rebaudioside D was silanised as described for the hapten R₄,using as silane (3-propilisocianate) triethoxysilane to obtain RebDhapten R₅ (FIG. 6). After 3 hours at room temperature, 5 mg of haptenwere added to a protein solution (10 mg of KLH, BSA, OVA and Hb in 5 mLof 10 mM PBS or 3 mg of HRP in 1.5 mL of PBS), leaving the reaction 2hours at room temperature. In this case the conjugates were also usedwithout purification.

EXAMPLE II Immunization Schedule and Antiserum Preparation

The hapten R₁ (rebaudioside D) (5 mg suspended in 0.5 mL of 10 mM PBS)and KLH-conjugates or BSA-R (n=2-5), (0.5 mL of conjugate at aconcentration of 200 mg/mL, 10 mM PBS) were injected intramuscularlyinto two New Zealand California white rabbits, with 0.5 mL of completeFreund's adjuvant in the first immunization, and 0.5 mL of incompleteFreund's adjuvant in the following. Immunizations were performed every21 days, and 10 days after each immunization bleedings were carried outto know the title and properties of serum from each animal. After fivecycles of immunization, blood was obtained by bleeding, leaving it tocoagulate overnight at 4° C. Subsequently, after adding sodium azide(0.02%) serum was separated by centrifugation and stored at −80° C.

Sera obtained were R₁ (I and II), BSA-R₂ (I and II), KLH-R₂ (I and II),BSA-R₃ (I and II), KLH-R₃ (I and II), BSA-R₄ (I and II), KLH-R₄ (I andII), BSA-R₅ (I and II) and KLH-R₅ (I and II). Symbols I and II, denotetwo white rabbits immunised for each immunogen.

When the schedule was completed, whole blood was collected and allowedto coagulate overnight at 4° C. Then serum was separated bycentrifugation and aliquots of sera were stored at 4° C. in 50% ammoniumsulphate.

EXAMPLE III Screening of Sera and Coating Conjugate

The titre of the antisera was determined by checkerboard titrationassay, using a non-competitive indirect ELISA.

First, the avidity of the sera obtained against different coatingconjugates was determined by non-competitive indirect ELISA usingdifferent antisera (serial dilutions from 1/1000 to 1/64000) andconcentrations of the coating conjugates (0.001 to 1.0 mg/L). Similarly,the avidity to the enzyme-tracer was determined by a directnon-competitive ELISA using several dilutions of the tracer (serialdilutions from 0.001 to 1.0 mg/L) and serum (from 1/500 to 1/512000).

The titres obtained from conjugate-coated format indirect format (ELISAplates coated with protein-hapten) were higher than those obtained fordirect format (ELISA plates coated with serum), choosing as optimalconcentrations of coating conjugates and dilution of serum, those thatproduced absorbance values around 0.8-1.2 units in absence of analyte(Rebaudioside D), to carry out competitive tests.

Serum KLH-R₃ (I) showed competition with several coating conjugates. So,the combination KLH-R₃ (I)/BSA-R₃ (1:2000/0.03 mg/L) was selected, sincepresented the lowest IC₅₀ value (1.30 μg/L) to establish an ELISA forRebaudioside D.

EXAMPLE IV Optimization and Performance of ELISA Assay for RebaudiosideD IV.1. Conjugate-Coated Format

Flat-bottomed polystyrene ELISA plates were coated overnight at 4° C.with 100 μL/well of the appropriate coating conjugate solution in CB.The following day, plates were washed six times with 15 mmol/L PBS-T pH8.5, and then 50 μL of the serum dilution and 50 μL of Reb D in PBS-Twere added and incubated at room temperature during 1 h. After washing,an incubation step (1 h) with 100 μL/well GAR-HRP (diluted 1/2000 inPBS-T) was accomplished, and plates washed again. Finally, 100 μL/wellof substrate solution (2 mg/mL OPD and 0.012% H₂O₂ in 25 mmol/L sodiumcitrate and 62 mmol/L sodium phosphate, pH 5.5) was added. The enzymaticreaction was stopped after 10 min by addition of 2.5 mol/L H₂SO₄ (50μL/well), and the absorbance was read in a dual-wavelength mode (490,650 nm).

IV.2. ELISA Optimization

The optimization was performed for the most sensitive assay (antiserumKLH-R₃/BSA-R₃ coating conjugate) using Reb D as competitor analyte.

The influence of several experimental parameters such as ionic strength,pH, surfactant concentration and time of competition on assaycharacteristics was examined in order to improve the immunoassayperformance (sensitivity, maximum absorbance, working or dynamic rangeand detection limit). Criteria used to evaluate the assay performanceswere sensitivity (IC₅₀), maximum absorbance (A₀), dynamic range (DR,established between the Reb D concentrations producing 20% and 80%colour inhibition) and limit of detection (LD, estimated at 10%inhibitory concentration).

IV.3. Effect of Ionic Strength

To study the influence of ionic strength, different tests were performedvarying the concentration of PBS in the competition buffer, between 5and 30 mM, 0.05% (v/v) Tween 20, pH 7.5. The maximum signalsignificantly increased when ionic strength decrease, while thesensitivity (IC₅₀) was clearly improved at 15 mM, so this was theselected value of the buffer.

IV.4. Effect of pH

To study the effect of pH on the assay sensitivity and maximum signal,15 mM, 0.05% (v/v) Tween 20 competition buffer solutions were preparedat different pH (4.5, 5.5, 6.5, 7.5, 8.5 and 9.5). It was observed thatthe best sensitivity was obtained at pH 8.5.

IV.5. Effect of Surfactant Concentration

The effect of surfactant concentration was studied with differentsolutions of PBS competition buffer, using the conditions previouslyoptimised (ionic strength and pH), varying the concentration of Tween 20(0 to 0.25%). The best IC₅₀ value was obtained with 0.25% Tween 20.Thus, the optimal parameters for the Reb D immunoassay developed were:15 mM PBS, pH 8.5, and 0.25% Tween 20 (see Table 1).

TABLE 1 Effect of different parameters on the ELISA performanceParameter IC₅₀ (μg/L) A₀ (Absorbance Units) PBS (mmol/L) 5 1.53 ± 0.412.39 ± 0.22 10 1.30 ± 0.35 1.73 ± 0.17 15 0.77 ± 0.24 1.49 ± 0.15 201.02 ± 0.28 1.22 ± 0.13 30 1.70 ± 0.45 1.06 ± 0.09 pH 4.5 0.63 ± 0.211.19 ± 0.08 5.5 0.86 ± 0.23 1.29 ± 0.10 6.5 0.91 ± 0.27 1.42 ± 0.14 7.50.77 ± 0.24 1.49 ± 0.15 8.5 0.49 ± 0.15 1.44 ± 0.12 9.5 0.50 ± 0.19 1.21± 0.09 Tween 20 (%) 0.000 0.68 ± 0.26 1.91 ± 0.21 0.010 0.56 ± 0.23 1.59± 0.19 0.025 0.58 ± 0.19 1.51 ± 0.18 0.050 0.49 ± 0.15 1.44 ± 0.12 0.1000.39 ± 0.08 1.20 ± 0.10 0.250 0.36 ± 0.05 1.15 ± 0.06 Pair KLH-R₃(I)/BSA-R₃ (1:2000/0.03 mg/L), PBS-T 15 mmol/L, 0.25% (v/v) Tween 20, pH8.5, 1 h competition, [GAR-HRP] 1/2000.

IV.6. Performance Under Optimal Conditions

After the optimization was performed, a set of 20 calibration curveswere carried out as shown in FIG. 8, which showed an IC₅₀ value of0.36±0.05 μg/L, a detection limit of 0.01 μg/L, and a dynamic range of0.04 to 1.91 μg/L for Reb D.

IV.7. Cross-Reactivity Studies

Assay selectivity was evaluated by determining the cross-reactivity withtwo sugars derived from Stevia rebaudiana, such as rebaudioside A andstevioside. The cross reactivity values were calculated according to thefollowing equation: CR=(IC₅₀ [mg/L] Rebaudioside D/IC₅₀ [mg/L]compound)×100.

The interferences observed were negligible for the two compounds tested(CR 0.16% with Rebaudioside A and 8.6×10⁻⁵% for Stevioside). Thus, thedeveloped immunoassay for Reb D is specific (maximum CR<10%) againstdifferent related compounds: Stevioside (IC₅₀=418,605 μg/L) and Reb A(IC₅₀=225 μg/L), which the highest interference was obtained (0.16%).

EXAMPLE V Analysis of Real Samples

Three different Stevia samples were extracted by two methods and theextracts analysed by HPLC and the developed ELISA.

V.1. Sample Extraction Procedure

For method one, the leaves was dried in a vacuum oven at 40° C. untilconstant weight (step 1). The dried leaves were grinded to fine powderusing laboratory grinder or mill (step 2). Approx. 0.5 g of fine powderswere placed in a centrifuge tube, then approx. 10 mL of water were addedinto the centrifuge tube, and the centrifuge tube was incubated in ashaking water bath at 55° C. for 1 h (step 3). The liquid (supernatant)was separated by centrifugation at 10,000 rpm for 15 min (step 4). Thesupernatant was filtered through filter paper into 50 mL volumetricflask (step 5). The biomass retained on the filter was returned to thecentrifuge tube and subjected to another round of extraction, separationand filtration as described in steps 3-5. In total 5 consecutiveextractions were carried out and the filtrates yielded from allextractions were mixed together (step 6). The volume of mixture wasadjusted to 50 mL with water. Then the obtained mixture was filteredthrough 0.2 μm syringe filter into HPLC vial and cap (step 7). Duplicatesamples were prepared following steps 3 to 7 (step 8).

For method two, 400 mg of crushed stevia leaf samples were placed in anopaque vial, containing 4 mL of acetonitrile:water (50:50). Aftervigorous agitation, the vial was placed in an ultrasonic bath for 10min. After that, samples were centrifuged at 15.000 rpm, using the upperphase for analysis.

The extracts obtained from both methods were then properly diluted(1/200, 1/400, 1/600 and 1/1000) (v/v) in PBS 2× and checked forRebaudioside D with the ELISA described in Example IV. In order toassess assay reproducibility, triplicates of each fortification levelwere performed. The mean value was compared to HPLC results, and percentrecovery calculated. The results are shown in Table 2.

The Rebaudioside D results obtained by HPLC were considered as realvalues (gold standard) in the leaf samples tested. In this sense,percent recovery (% R) was calculated as (mean ELISA results/HPLCresults)×100%.

It is understood that the results of Table 2 are illustrative of oneembodiment of ELISA using polyclonal antibody KLH-R3 (I) and BSA-R3 ascompetitor conjugate. Other competitive assays within the scope of thisinvention may provide different performance characteristics.

TABLE 2 Results for Reb D obtained for leaf extracts applying twoextraction and detection methods Sample 1 Sample 2 Sample 3 Method oneMethod two Method one Method two Method one Method two HPLC ELISA HPLCELISA HPLC ELISA HPLC ELISA HPLC ELISA HPLC ELISA 8.23 6.30 7.02 7.2110.61 10.00 12.81 12.86 5 6.18 3.43 3.74 (% R) = 76.5% (% R) = 102.7% (%R) = 94.2% (% R) = 100.4% (% R) = 126.6% (% R) = 109%

While the present invention has been described with reference toparticular embodiments, it will be understood that the embodiments areillustrative and that the invention scope is not so limited. Alternativeembodiments of the present invention will become apparent to thosehaving ordinary skill in the art to which the present inventionpertains. Such alternate embodiments are considered to be encompassedwithin the scope of the present invention. Accordingly, the scope of thepresent invention is defined by the appended claims and is supported bythe foregoing description.

1. A hapten derivative comprising a natural high intensity sweetenerderivatized with an imidazole carboxylic ester, said hapten derivativehaving the structure (R3):

wherein the “wavy line” indicates rest of the structure of the naturalhigh intensity sweetener, which in combination with the disclosed sugarmoiety in (R3) makes the full structure of the natural high intensitysweetener, and wherein the hapten derivative is used for further makingconjugates for being used as an immunogen for generating antibodiesspecific for the natural high intensity sweetener or a reagent fordetecting the natural high intensity sweetener, wherein the natural highintensity sweetener comprises steviol glucosides.
 2. The haptenderivative of claim 1, wherein the natural high intensity sweetener isselected from the group consisting of Dulcoside A, Rebaudiosides A, B,C, D, E, and F, steviolbioside, Rubusoside, and combinations thereof. 3.(canceled)
 4. The hapten derivative of claim 1, wherein the R3 is formedby reacting the natural high intensity sweetener withN,N′-carbonyldiimidazole. 5-21. (canceled)